|Year : 2017 | Volume
| Issue : 2 | Page : 208-214
Botanical and chemical fingerprinting of medicinal roots of Justicia gendarussa burm f.
Deepa Yadav1, Mohd Salim Reshi1, Chhavi Uthra1, Sadhana Shrivastava1, Nalini Srivastava2, Sunil Kumar Koppala Narayana3, Sangeeta Shukla1
1 UNESCO- Trace Element Satellite Center, School of Studies in Zoology, Gwalior, Madhya Pradesh, India
2 School of Studies in Biochemistry, Jiwaji University, Gwalior, Madhya Pradesh, India
3 SDM Center of Research in Ayurveda and Allied Sciences, Laxminarayana Nagar, Kuthpady, Udupi, Karnataka; Department of Pharmacognosy, Siddha Central Research Institute, Arumbakkam, Chennai, India
|Date of Web Publication||18-Apr-2017|
Prof. Sangeeta Shukla
Jiwaji University, Gwalior - 474 011, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Justicia gendarussa Burm f. of family Acanthaceae is medicinally important herb used in the treatment of inflammatory disorders, asthma, hepatic injuries, pathogenic infection and also shows antiproliferative activity against various cancer cell lines. Materials and Methods: Pharmacognostical evaluation (macro-microscopy, physicochemical analysis and preliminary phytochemical analysis), high-performance thin layer chromatography (HPTLC) fingerprinting and chemical profiling by gas chromatography-mass spectrometry (GCMS) of dried roots of J. gendarussa were done according to quality standard procedures. Results: Microscopic analysis revealed the compact arrangement of cells in cork region and thin-walled cortex beneath epidermis. Parenchymatous cells with xylem vessel were observed in the roots of J. gendarussa. Physicochemical studies revealed loss on drying (10.474%), total ash (2.990%), acid-insoluble ash (0.099%), water-soluble ash (1.528%), alcohol-soluble extractive value (0.564%) and water-soluble extractive value (4.11%) of the raw drug. Preliminary phytochemical analysis of ethanolic extract of J. gendarussa showed the presence of alkaloid, steroid, flavonoid, phenol, carbohydrate, saponin and quinone. Rf, color of the spots and densitometric scan were recorded by HPTLC fingerprinting using toluene: ethyl acetate: formic acid (5.0:4.0:1.0). On photodocumentation, six spots were visualized under 254 nm, nine spots under 360 nm and six spots at 620 nm. Identification of components in ethanolic extract of J. gendarussa was done by GC-MS. GC-MS results in the presence of oleic acid, 9,12-octadecadienoic acid, 6,9,12-octadecatrienoic acid and estra-1, 3,5 (10)-trein-17-β-ol in ethanolic extract of J. gendarussa. Conclusion: These specific identities will be useful in identification and authentication of the raw drug in dried form.
Keywords: Gas chromatography-mass specrtometry analysis, high-performance thin layer chromatography fingerprinting, Justicia gendarussa, pharmacognostic, quality control
|How to cite this article:|
Yadav D, Reshi MS, Uthra C, Shrivastava S, Srivastava N, Narayana SK, Shukla S. Botanical and chemical fingerprinting of medicinal roots of Justicia gendarussa burm f. Phcog Res 2017;9:208-14
|How to cite this URL:|
Yadav D, Reshi MS, Uthra C, Shrivastava S, Srivastava N, Narayana SK, Shukla S. Botanical and chemical fingerprinting of medicinal roots of Justicia gendarussa burm f. Phcog Res [serial online] 2017 [cited 2020 Jan 24];9:208-14. Available from: http://www.phcogres.com/text.asp?2017/9/2/208/204643
- Transverse section and powder of dried roots of Justicia gendarussa were examined microscopically. Microscopic observations showed the presence of well.developed cork and cortex. Presence of xylem vessels and parenchymatic rays were observed in transverse section. Parenchymatous cell and sclereids with vessel elements were found in powder microscopy
- Physicochemical studies revealed loss on drying (10.474%), total ash (2.990%), acid-insoluble ash (0.099%), water-soluble ash (1.528%), alcohol-soluble extract (0.564%) and water-soluble extract (4.11%)
- Preliminary phytochemical analysis of ethanolic extract of J. gendarussa showed the presence of alkaloid, steroid, flavonoid, phenol, carbohydrate, saponin and quinone
- High.performance thin layer chromatography fingerprinting showed different peaks at different wavelength
- Chemical profiling of medicinal roots of J. gendarussa by gas chromatography. mass spectrometry revealed the presence of oleic acid, 9,12.octadecadienoic acid, 6, 9, 12.octadecatrienoic acid and estra-1, 3, 5 (10)-trein-17-β-ol as bioactive compound.
Abbreviations Used: TLC: Thin layer chromatography; HPTLC: High performance thin layer chromatography; GCMS: Gas chromatography-mass specrtometry; QSIMP: Quality standard of indian medicinal plant; LOD: Loss on drying; TA: Total ash; AIA: Acid insoluble ash; WSA: Water soluble ash; ASE: Alcohol soluble extractive; WSE: Water soluble extractive.
| Introduction|| |
J. gendarussa Burm f. Syn. Gendarussa vulgaris Nees is shade friendly, rapidly escalating and fragrant herb grown in India. J. gendarussa is commonly known as Nili nirgunthi and Krishna nirgundi in Hindi, Bakas and Kala adulsa in Marathi, Kasanah and Vaidhyasinha in Sanskrit, and Karunochi in Tamil.,, It is an erect, branched and smooth herb about one meter in height, leaves of J. gendarussa are linear-lanceolate and glabrous in appearance and flowers are small, white with pink or purple spots inside [Figure 1]a and [Figure 1]b.
|Figure 1: (a-e) Macroscopic features of Justicia gendarussa. (f-k) Microscopic observations of dried roots of Justicia gendarussa. Phloem (Ph); Cork (Ck); Vessel (Ve); Xylem Rays (XR); Phloem fibres (PF); Cortex (CO); Pith (Pi)|
Click here to view
J. gendarussa is traditionally used in the treatment of chronic rheumatism, inflammations, bronchitis, eye diseases, fever, headache, earache, muscle pain, respiratory disorder and digestion problems.,,,, Roots of J. gendarussa are known to possess antipyretic, antiangiogenic, antimicrobial, antinociceptive and antiproliferative activity. Previous studies on J. gendarussa revealed the presence of active phytoconstituents such as flavonoids, alkaloids, triterpenoid saponins, amino acids, aromatic amines, stigmasterol and lupeol which help in reducing the oxidative stress.,,,,,
Ethanolic extract of this plant showed a significant antiarthritic activity against Freund's adjuvant-induced and collagen-induced arthritic rat models. Leaves and stem of J. gendarussa have been reported for anthelmintic activity and antibacterial activity against Staphylococcus aureus, Escherichia More Details coli, Pseudomonas aeruginosa and Vibrio cholera.,,, Methanolic extract of leaves of J. gendarussa is reported for its cytotoxic effect against some human cancer cell lines and also helps in ameliorating the CCl4-induced hepatic injury.,, Earlier findings of expertise validate that methanolic extract of its roots possess anti-inflammatory potential against carrageenan-induced inflammation and an ethyl acetate fraction isolated from methanolic extract of roots of J. gendarussa showed the anti-inflammatory effect by inhibiting the expression of iNOS and COX-2 through NF-κB pathway.
The present study was designed to prepare a complete monograph for standardization and authentication of roots of J. gendarussa in dried form as this plant is still untouched drug in Ayurveda Pharmacopoeia of India and Quality Standards of Indian Medicinal Plants.
| Materials and Methods|| |
Macroscopic characters of dried roots and powder were keenly observed under naked eyes to record the specific botanical characters. The external features of the test samples were documented using Canon IXUS digital camera.
Dried roots were preserved in formalin-acetic acid-alcohol preservative solution (5% formalin - 5 ml, 5% acetic acid - 5 ml and 50% ethyl alcohol - 90 ml). After 48 h, very thin transverse sections of root were obtained using sharp blade followed by safranin staining  for microscopic visualization. Features were photographed using Zeiss AXIO trinocular microscope attached with Zeiss AxioCam camera under bright field light. Magnifications of figures are indicated in scale bars.
A pinch of coarse powder sifted through 80 pore size mess was mixed with drops of choral hydrate on microscopic slides and mounted with a drop of glycerin-water. Slides were observed and characterized under Zeiss AXIO trinocular microscope. Magnifications are indicated by scale bars.
Physicochemical characterization such as loss on drying (LOD) at 105°C, total ash (TA), acid-insoluble ash (AIA), water-soluble ash (WSA), alcohol-soluble extractive (ASE) value (ASE) and water-soluble extractive (WSE) value were determined as per Quality Standard of Indian Medicinal Plants.
Preliminary phytoconstituents screening
Preliminary phytoconstituents screening was done to detect the presence of active constituents in the ethanolic extract of J. gendarussa.
High-performance thin layer chromatography fingerprinting
One gram of powdered roots was extracted with 10 ml ethanol and kept for cold percolation for 24 h and filtered. 4, 8 and 12 μl of the plant extract were applied on a precoated silica gel F254 on aluminum plates to a bandwidth of 7 mm using Linomat 5 (CAMAG, Muttenz, Switzerland) TLC applicator. J. gendarussa plate was developed using toluene:ethyl acetate:formic acid (5.0:4.0:1.0) as mobile phase in CAMAG twin trough chamber. The developed plate was visualized under short UV, long UV in CAMAG TLC photodocumentation unit, then derivatized with anisaldehyde-sulfuric acid reagent, and scanned under UV 254, 366 and 620 nm postderivatisation. Rf, color of the spots and densitometric scan were recorded using CAMAG Scanner 4.,
Gas chromatography-mass specrtometry analysis
Gas chromatography-mass specrtometry (GC-MS) analysis was carried out using Thermo Scientific Mass Spectrophotometer equipped with Triple Quad XLS. The column used was HP-5ms Ultra Inert (length: 30.0 m; diameter: 0.25 mm), with a film thickness of 0.25 μm. The carrier gas used was helium at a flow rate of 1.3 ml/min at a constant rate. Two microliters sample injection volume was utilized. The inlet temperature was maintained as 280°C. The oven temperature was programed initially at 60°C for 3.5 min and then programed to increase to 300°C at a rate of 10°C. Total run time was 22 min. The MS transfer line was maintained at a temperature of 240°C. MS was recorded using electron impact at fixed electron energy of 70eV and data were evaluated using total ion count for compound identification and quantification. The spectra of the components were compared with the spectral database of known components in the GC-MS library (NIST-11). Measurement of peak areas and data processing were carried out by Turbo-Mass-OCPTVS-Demo SPL software.
| Results|| |
Macroscopically, the dried roots of J. gendarussa were about 10 cm long with a diameter of 0.5 cm. Dried roots were yellowish brown in color with rough and wrinkled surface and root scars. Powder of root was yellowish in color [Figure 1]c,[Figure 1]d,[Figure 1]e with pleasant odor. Transverse section of dried root showed elongated, compactly arranged cork cell and cortex of root was well developed under epidermis. Outer and inner region of root showed the presence of xylem vessels with some intracellular spaces. Xylem vessels were spherical and oval in shape. Large xylem vessels were found toward the outer region and their size was gradually decreased toward the inner region near pith. Parenchymatic rays were arranged in a uniseriate manner [Figure 1]f,[Figure 1]g,[Figure 1]h,[Figure 1]i,[Figure 1]j,[Figure 1]k. Powder microscopy showed the presence of pitted lignified parenchymatous cells with lobed projection, sclereids of various dimensions were scattered and fiber sclereids with vessel elements were found. Group of stone cells were also observed in powder microscopy [Figure 2]a,[Figure 2]b,[Figure 2]c,[Figure 2]d,[Figure 2]e,[Figure 2]f,[Figure 2]g,[Figure 2]h,[Figure 2]i,[Figure 2]j,[Figure 2]k,[Figure 2]l.
Physicochemical characters such as LOD, TA value, AIA, water-insoluble ash, ASE and WSE are expressed in %w/w [Table 1].
Preliminary phytochemical analysis
According to Harborne's methodology, phytoconstituents analysis revealed the presence of carbohydrates and some secondary metabolites such as alkaloids, steroids, flavonoids, phenols, saponins and quinone [Table 2].
High-performance thin layer chromatography fingerprinting
Rf values and color of the spots in chromatogram developed in toluene: ethyl acetate:formic acid (5.0:4.0:1.0) for ethanolic extract of dried roots were recorded [Table 3]. TLC photodocumentation revealed the presence of many phytoconstituents at different Rf values. High-performance thin layer chromatography (HPTLC) densitometric scan of the plates showed numerous bands under short UV, long UV and 620 nm (after derivatization). On photodocumentation, under short UV, six spots were observed; under long UV, there were nine spots and under 620 nm on postderivatization with anisaldehyde-sulfuric acid spray reagent, six spots were recorded [Figure 3]a,[Figure 3]b,[Figure 3]c. Densitometric scan at 254 nm revealed ten peaks corresponding to ten different compounds in the ethanolic extract, compounds with Rf0.04 (40.01%), 0.17 (11.99%), 0.23 (26.47%), 0.40 (1.95%), 0.42 (1.71%), 0.53 (0.93%), 0.62 (9.59%), 0.67 (1.77%), 0.80 (1.96%) and 0.90 (3.62%) are shown in [Figure 4]a. Densitometric scan at 366 nm [Figure 4]b showed six peaks such as Rf - 0.05 (3.71%), 0.15 (4.05%), 0.25 (2.20%), 0.58 (5.97%), 0.64 (2.61%) and 0.96 (81.46%). [Figure 4]c depicts seven peaks with Rf - 0.03 (23.91%), 0.23 (22.20%), 0.26 (14.84%), 0.48 (2.98%), 0.72 (6.91%), 0.81 (26.22%), and 0.86 (2.93%) after postderivatization at 620 nm.
|Figure 3: (a-c) High-performance thin layer chromatography photodocumentation of ethanolic extract of Justicia gendarussa|
Click here to view
|Figure 4: (a-c) HPTLC densitometric scan of ethanolic extract of Justicia gendarussa|
Click here to view
Gas chromatography-mass specrtometry analysis
GC-MS of J. gendarussa indicated the presence of five constituents. Out of five constituents, one was unidentified. The remaining four constituents were found in trace amounts [Table 4]. Compounds identified from GC-MS analysis were fatty acids (oleic acid, 9,12-octadecadienoic acid and 6, 9, 12-octadecatrienoic acid) and steroids (estra-1, 3, 5(10)-trein-17-β-ol), which correlate well with the results of phytochemical screening. Mass spectrum of ethanolic extract of J. gendarussa indicated the similarity of identified compounds and structures with different retention times as expressed in [Figure 5]a,[Figure 5]b,[Figure 5]c,[Figure 5]d and [Figure 6].
|Table 4: Details of compounds identified from ethanolic extract of Justicia gendarussa Burm F. root|
Click here to view
|Figure 5: (a-d) Mass specrtometry of compounds identified from ethanolic extract of Justicia gendarussa root|
Click here to view
|Figure 6: Gas-liquid chromatogram of ethanolic extract of Justicia gendarussa|
Click here to view
| Discussion|| |
Pharmacognostic analysis with physicochemical studies and HPTLC fingerprinting was done for authentication and quality control of drug. Macro-microscopic characters showed the presence of compactly arranged cork cell and cortex. Transverse section of root showed the presence of xylem vessels in a different shape. Gradually decreased xylem vessels were found. Parenchymatic cells with lobed projection, sclereids, fibers and group of stone cells were also observed in powder microscopy. The findings of the present study were supported by other expertise.
 The physicochemical constants of J. gendarussa were standardized to check for purity of drug., Deterioration time of drug depends on its water contents as LOD at 105°C was 10.474%. TA (2.990%) represents the inorganic residue after incineration. AIA percentage reveals the presence of siliceous substances in drug. By treating the TA with dilute hydrochloric acid, the percentage of AIA was determined; (0.099%) minimum AIA value percentage means less contamination with siliceous matter while the WSA (1.528%) indicates the inorganic contents after treatment of the TA with water. Secondary metabolites of plants are intended for their therapeutic values are extracted in suitable solvents (water and alcohol). The ASE values (0.564%) support the presence of polar components of the plant such as alkaloids, steroids, flavonoids and glycosides whereas the WSE value (4.11%) represents the presence of sugar and acids. HPTLC as quality assessment tool for the identification of variation in chemical constituents showed different Rf at different wavelengths. Values as Rf at different wavelength under short UV, long UV and after postderivatization can serve as quality fingerprint for roots of J. gendarussa. GC-MS is the most commonly used technique for the identification and quantification purpose. Active constituents of plants material can be determined by GC-MS analysis and data interpretation can be done by matching the spectra with mass spectrum library such as NIST. GC-MS of ethanolic extract of dried rhizomes revealed the presence of four compounds out of which oleic acid is reported to induce apoptosis in carcinoma cells by increasing the intracellular ROS production or caspase-3 activity  and 9,12-octadecadienoic acid (linoleic acid) is reported to possess anti-inflammatory, nematicide, insectifuge, hypocholesterolemic, anticancer, hepatoprotective, antihistaminic, antiacne, antiarthritic and antieczemic activity. GC-MS results of ethanolic extract of dried roots showed the presence of pharmacologically active components.
| Conclusion|| |
Macro-microscopic observations, physicochemical analysis, preliminary phytochemical screening and HPTLC fingerprinting could be utilized as reference limits for the quality control standards to study the roots of J. gendarussa. Chemical profiling using GC-MS revealed the presence of omega fatty acids and sterol in ethanolic extract of the dried roots of J. gendarussa. The data obtained from the study can be used to study the therapeutic efficacy of compounds on the pharmacological activity.
Authors are highly grateful to Jiwaji University, Gwalior, Madhya Pradesh. Authors highly regard Dr. B. Ravishankar, Director, SDM Centre for Research in Ayurveda, for providing the laboratory facilities. Suchitra N. Prabhu is gratefully acknowledged for constant support during the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Handa SS, Sharma A, Chakraborti KK. Evaluation of Indian herbal hepatoprotective drugs. Fitoterapia 1986;57:307-51.
Krishna KL, Mruthunjaya K, Patel JA. Antioxidant and hepatoprotective activity of stem methanolic extract of Justicia gendarussa
Burm. Int J Pharmacol 2010;6:72-80.
Janarthanam B, Sumathi E.In vitro
regeneration of Justicia gendarussa
Burm. F. Libyan Agric Res J Int 2010;1:284-7.
Khatijah H, Noraini T. Anatomical Atlas of Medicinal Plants. Vol. 1. Penerbit UKM; 2007. p. 106.
Ayob Z, Samad AA, Bohari SP. Cytotoxicity activities in local Justicia gendarussa crude extracts against human cancer cell lines. J Teknologi (Sci Eng) 2013;64:45-52.
Anonymous. The Wealth of India: Raw Materials. New Delhi: Council of Scientific and Industrial Research; 1959. p. 312.
Thomas TD, Yoichiro H.In vitro
propagation for the conservation of a rare medicinal plant Justicia gendarussa
Burm. F. by nodal explants and shoot regeneration from callus. Acta Physiol Plant 2010;32:943-50.
Kirtikar CR, Basu BD. Indian Medicinal Plants. Allahabad: F. L. M. Basu; 1935. p. 1896-8.
Ratnasooriya WD, Deraniyagala SA, Dehigaspitiya DC. Antinociceptive activity and toxicological study of aqueous leaf extract of Justicia gendarussa
Burm. F. in rats. Pharmacogn Mag 2007;3:145-55.
Periyanayagam K, Umamaheswari B, Suseela L, Padmini M, Ismail M. Evaluation of antiangiogenic effect of the leaves of Justicia gendarussa
(Burm F) (Acanthaceae
) by chrio allontoic membrane method. Am J Infect Dis 2009;5:180-2.
Chakravarty AK, Dastidar PP, Pakrashi SC. Simple aromatic amines from Justicia gendarussa. 13
CNMR spectra of the bases and their analogues. Tetrahedron 1982;38:1797-802.
Krishna K, Mruthunjaya K, Patel JA. Antioxidant and hepatoprotective activity of leaves extract of Justicia gendarussa
Burm. Int J Biol Chem 2009;3:99-100.
Mustafa RA, Hamid AA, Mohamed S, Bakar FA. Total phenolic compounds, flavanoids and radical scavenging activity of 21 selected tropical plants. J Food Sci 2010;75:28-35.
Paval J, Kaitheri SK, Potu BK, Govindan S, Kumar RS, Narayanan SN, et al.
Anti-arthritic potential of the plant Justicia gendarussa
Burm F. Clinics (Sao Paulo) 2009;64:357-62.
Saha MR, Debnath PC, Rahman MA, Islam MA. Evaluation of in vitro
anthelmintic activities of leaf and stem extracts of Justicia gendarussa
. Bangladesh J Pharmacol 2012;7:50-3.
Subrahmanian N, Jothimanivannan C, Moorthy K. Antimicrobial activity and preliminary phytochemical screening of Justicia gendarussa
(Burm F.) against human pathogens. Asian J Pharm Clin Res 2012;5:229-33.
Sudhanandh VS, Arjun JK, Faisal AK, Ani MV, Renjini VS, Narendra BK. In-vitro antibacterial screening of selected folklore Indian medicinal plants with few clinical pathogens. Indian J Pharm Educ Res 2012;46:174-8.
Sivasakthi A, Vijayalakshmi M. Antibacterial activities of phytochemical extracts from the leaves of Justicia gendarussa
Burm F. Int J Pharm Bio Sci 2014;5:433-8.
Ayob Z, Wagiran A, Abd Samad A. Potential of tissue cultured medicinal plants in Malaysia. J Teknologi 2013;62:111-7.
Ayob Z, Mohd Bohari SP, Abd Samad A, Jamil S. Cytotoxic activities against breast cancer cells of local Justicia gendarussa
crude extracts. Evid Based Complement Alternat Med 2014;2014:732980.
Phukan B, Kakoti BB, Verma VK, Kumar A. Hepatoprotective activity of Justicia gendarusa
Linn. leaves in carbon tetrachloride induced liver injury in mice. J Nat Remedies 2014;14:132-7.
Kumar SK, Vijayan V, Bhaskar S, Krishnan K, Shalini V, Helen A. Antiinflammatory potential of an ethyl acetate fraction isolated from Justicia gendarussa
roots through inhibition of iNOS and COX-2 expression via NF-κB pathway. Cell Immunol 2012;272:283-9.
Sass JE. Elements of Botanical Micro Technique. New York: McGraw Hill Book Co.; 1940.
O'Brein TP, Feder N, McCull ME. Polychromatic staining of plant cell walls by toluidine blue-O. Protoplasma 1964;59:368-73.
Yadav D, Reshi RM, Shrivastava S, Srivastava N, Kumar SK, Shukla S. Macro-microscopic evaluation, physicochemical analysis and HPTLC finger printing of Curculigo orchioides
Gaertn. Rhizome. Pharmacogn J 2016;8:1-5.
Quality Standard of Indian Medicinal Plant, Indian Council of Medical Research, New Delhi; 2014.
Harborne JB. Phytochemical Methods. 2nd
ed. London: Chapman and Hall Ltd.; 1973. p. 42.
Waksmundzka HM, Sherma J, Kowalska T. Thin Layer Chromatography in Phytochemistry. London: CRC Press, Taylor and Francis Group; 2008. p. 595.
Stahl I. Thin Layer Chromatography. A Laboratory Hand Book (Student Edition). Berlin: Springer-Verlag; 1969. p. 52-86.
Sethi PD. High Performance Thin Layer Chromatography. 1st
ed. New Delhi: CBS Publishers and Distributors; 1996.
Prasad AD, Prasad AB, Prasad KD, Shetty P, Kumar SK. GCMS compositional analysis of essential oil of leaf and fruit rind of Citrus maxima (Burm.) Merr. from Coastal Karnataka, India. J Appl Pharm Sci 2016;6:68-72.
Patel S, Zaveri M. Pharmacognostic study of the root of Justicia gendarussa
Burm. Asian J Tradit Med 2011;6:61-72.
The Ayurvedic Pharmacopoeia of India. Part III. 1st
ed. Delhi: Controller of Publications Civil Lines; 2008. p. 153-64.
Kumar SK, Saraswathy A, Amerjothy S. HPTLC fingerprinting of extracts of mango mistletoe Helicanthus elastica
(Desr.) Danser with multiple markers. J Sci Innov Res 2013;2:864-71.
Carrillo C, Cavia Mdel M, Alonso-Torre SR. Antitumor effect of oleic acid; mechanisms of action: a review. Nutr Hosp 2012;27:1860-5.
Sermakkani M, Thangapandian V. GC-MS analysis of Cassia italica
leaf methanol extract. Asian J Pharm Clin Res 2012;5:90-4.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]